利用甘蓝游离小孢子不定芽叶片再生DH植株技术研究

Study on plantlet regeneration technique of DH lines with leaf of adventitious buds from isolated microspore culture in cabbage(Brassica oleracea L.)

【目的】探索一种能将有限的DH株快速扩繁的植株再生技术,扩大DH株数量以提高育种效率。【方法】以甘蓝小孢子胚状体不定芽叶片为试材,MS为基础培养基,分别添加不同质量浓度的6-BA(1.0,2.0,3.0,4.0mg/L)和NAA(0.05,0.1,0.2,0.4mg/L),筛选出最优质量浓度组合;在6-BA和NAA最优质量浓度组合上,继续添加不同质量浓度的GA3(0.5,1.0,1.5,2.0mg/L),比较筛选小孢子不定芽叶片的最佳再生方法。最后,在MS+6-BA 2.0mg/L+NAA 0.1mg/L不定芽诱导培养基上,研究不同生长日龄DH株叶片的诱导效果。【结果】在MS+6-BA 2.0mg/L+NAA 0.1mg/L中,不定芽再生频率最高,为64.29%,分化系数4.03;添加6-BA、NAA和GA33种激素,对不定芽再生频率促进作用更大,其中MS+6-BA 2.0mg/L+NAA 0.1mg/L+GA31.5mg/L诱导效率最高,不定芽再生频率达到了80.36%,分化系数为4.15。当DH株叶片生长日龄为25d时,不定芽再生频率相对最大,达到68.76%,分化系数为3.62,35d后诱导效果下降。【结论】甘蓝DH株叶片诱导植株再生的适宜培养基为MS+6-BA 2.0mg/L+NAA 0.1mg/L+GA31.5mg/L;以生长日龄为25d左右的甘蓝DH株叶片作为外植体诱导植株再生效果较好。

【Objective】 We studied the high efficient plantlet regeneration method on the leaf of microspore culture plantlet in cabbage（Brassica oleracea L.） to get a great quantity of DH lines to promote the efficiency in cabbage breeding.【Method】 The leaves of DH lines＇ adventitious buds were inoculated on several types of MS-based media for the plantlet regeneration.The effects of combination of 6-BA（1.0,2.0,3.0,4.0 mg/L） and NAA（0.05,0.1,0.2,0.4 mg/L） on plantlet regeneration were studied,then GA3 was added in the selection of the optimum mixing concentration of 6-BA and NAA.By analysis and comparison,the best method was found.In addition,taking the age of leaf explants as the research object,explants were inoculated on MS＋6-BA 2.0 mg/L＋NAA 0.1 mg/L to compare the effect of the different growth periods on the leaf regeneration of DH lines.【Result】 In the media MS＋6-BA 2.0 mg/L＋NAA 0.1 mg/L,shoot regeneration frequency was the highest（64.29%）,differentiation coefficient was 4.03.Regeneration frequency was increased greatly after adding different concentrations of GA3 on MS＋6-BA 2.0 mg/L＋NAA 0.1 mg/L,MS ＋ 6-BA 2.0 mg/L＋NAA 0.1 mg/L＋GA3 1.5 mg/L was proved best,shoot regeneration frequency was 80.36%,differentiation coefficient 4.15.Shoot regeneration frequency reached its maximum value 68.76% when the age of leaf explants was 25 days,differentiation coefficient was 3.62,and it decreased sharply after 35 days.【Conclusion】 The suitable medium on adventitious bud regeneration of the leaf of DH lines is MS＋6-BA 2.0 mg/L＋NAA 0.1 mg/L＋GA3 1.5 mg/L.The age of leaf explants produces significant effect on the plantlet regeneration and differentiation,and 25-day-old leaf explants express the best proliferous effect.